The long term objective of this competing renewal grant application is to develop novel prodrug strategies to improve corneal permeability of topically applied antimicrobial agents employed in the treatment of both bacterial and viral corneal stromal and epithelial keratitis. In this grant proposal we aim to characterize the ascorbate carrier systems expressed on the corneal membrane. We also propose to synthesize amino acid, peptide and ascorbate ester prodrugs of erythromycin, levofloxacin and ofloxacin and ascorbate ester prodrugs of ACV, targeted towards the respective transporters expressed on the corneal membrane, and to evaluate their ability to circumvent P-gp and MRP mediated efflux. The specific aims of this renewal application are a) To synthesize derivatives of erythromycin, levofloxacin and ofloxacin targeting amino acid (phenylalanine and glutamate prodrugs), peptide (valine, valine-valine and glycine-valine prodrugs) and ascorbate (ascorbic acid prodrugs) transporters expressed on the cornea and to evaluate these conjugates with respect to their b) physiochemical properties and cytotoxicity; c) bio- reversion in ocular fluids and cell homogenates, d) uptake, transport and simultaneous bioreversion across isolated rabbit cornea, and human and rabbit primary corneal epithelial cell cultures, e) affinity for P-gp and MRP efflux pumps, f) in vitro antibacterial efficacy and g) in vivo antibacterial efficacy against bacterial keratitis in the rabbit model. In vitro and in vivo studies will be conducted by Dr. William Suling at the Southern Research Institute, Birmingham, AL and by Dr. James Hill at the LSU Eye Center, New Orleans, LA, respectively. Uptake and transport of erythromycin, levofloxacin and ofloxacin prodrugs will be studied across isolated rabbit cornea, human and rabbit primary corneal epithelial cell cultures (rPCEC), alone, in combination and in the presence of topically applied anti-inflammatory steroids (prednisolone, prednisone and 6-alpha-methylprednisolone). We propose to synthesize ascorbate prodrug of ACV study transport and simultaneous bio-reversion across isolated rabbit corneas, and human and rabbit primary corneal epithelial cell cultures; antiviral efficacy against in vitro viral screens of HSV-1, HSV-2, CMV, VZV and EBV and in vivo antiviral efficacy against HSV-1 (McKrae strain) induced acute epithelial and stromal keratitis in the rabbit model. In vitro and in vivo studies will be conducted by Dr. Sam Ananthan and Dr. Earl Kern under NIAID funded research contract and by Dr. James Hill. \n vivo ocular bioavailability of erythromycin, levofloxacin, ofloxacin and ACV and their prodrugs will be examined following topical application (alone, in combination or in the presence of prednisolone, prednisone and 6-alpha-methylprednisolone as inhibitors of corneal efflux pumps) in both anesthetized and conscious rabbit models by employing ocular microdialysis technique.